DNA sequence information represents the information required for gene organization and regulation of most life forms. Accordingly, the development of reliable methodology for sequencing DNA has contributed significantly to an understanding of gene structure and function.
Generally, strategies for determining the nucleotide sequence of DNA involve the generation of a DNA substrate i.e., DNA fragments suitable for sequencing a region of the DNA, enzymatic or chemical reactions, and analysis of DNA fragments that have been separated according to their lengths to yield sequence information. More specifically, to sequence a given region of DNA, labeled DNA fragments are typically generated in four separate reactions. In each of the four reactions, the DNA fragments typically have one fixed end and one end that terminates sequentially at each of the four nucleotide bases, respectively. The products of each reaction are fractionated by gel electropheresis on adjacent lanes of a polyacrylamide gel. As all of the nucleotides are represented among the four lanes, the sequence of a given region of DNA can be determined from the four "ladders" of DNA fragments. Presently, there are two general approaches available to generate ladders of DNA fragments for determination of the nucleotide base sequence of DNA of interest. One approach involves nucleotide specific chemical modification and cleavage reactions as described by Maxam and Gilbert, Meth. Enz., 65:499 (1980). A second approach, which involves primer extension reactions in the presence of nucleotide specific chain terminators as described by Sanger et al., Proc. Natl. Acad. Sci., 74:5463 (1977), is most commonly used for sequence determination.
The Maxam-Gilbert technique, supra, relies upon the base-specific chemical cleavage of the DNA to be sequenced. After the DNA substrate is end labeled, it is subjected to chemical reactions designed to cleave the DNA at positions adjacent to a given base or bases. The labeled DNA fragments will, therefore, have a common labeled terminus while the unlabeled termini will be defined by the positions of chemical cleavage. This results in the generation of DNA fragments which can be separated by gel electrophoresis and identified. Alternatively, unlabeled DNA fragments can be separated after complete restriction digestion and partial chemical cleavage of the DNA, and hybridized with probes homologous to a region near the region of the DNA to be sequenced. See, Church et al., Proc. Natl. Acad. Sci., 81:1991 (1984).
The Sanger method, supra, involves the enzymatic synthesis of a strand complementary to the DNA to be sequenced. Essentially, a labeled complementary strand of a cloned single-stranded DNA is synthesized utilizing an oligonucleotide primer to initiate synthesis and dideoxynucleotides to randomly terminate synthesis. The primer, which anneals to a primer binding site of vector DNA flanking the DNA to be sequenced, is extended by a DNA polymerase in the presence of labeled and unlabeled deoxynucleoside triphosphates. The resulting reaction products, which include a distribution of DNA fragments having primer-defined 5'termini and differing dideoxynucleotides at the 3'termini, are then separated by gel electrophoresis and the base sequence of the fragments are identified.
While numerous modifications and improvements to the strategies referred to above have been developed, most sequencing techniques require the presence of a known primer binding site for every 300 to 500 nucleotides to be sequenced either, for example, for initiation of DNA synthesis or for hybridization to different length DNA fragments having a common end. However, as such approaches utilize a "ladder" of DNA fragments containing the primer binding site (or its complement), the amount of sequence information that can be obtained is limited by the present inability to resolve DNA fragments greater than 500 nucleotides in length on sequencing gels.
Accordingly, methodology described by Guo and Wu, Nucleic Acids Res., 10:2065 (1982); and Meth. Enz., 100:60 (1983), which is not dependent upon primer binding sites, is highly desirable for sequencing DNA greater than 500 nucleotides. This method involves partially digesting linear double stranded DNA with E. coli exonuclease III to produce DNA fragments with 3' ends shortened to varying lengths, performing the dideoxy primer extension reactions of Sanger, supra, with the shortened 3' ends as primers for DNA synthesis, and digesting the DNA with a selected restriction enzyme that cleaves near one end of the molecule adjacent to, but not within, the labeled region of DNA. By digestion of the DNA with a selected restriction enzyme, the labeled DNA strands from one end of the molecule are made small enough to be resolved on a sequencing gel. Each successive deletion in length, therefore, brings "new" regions of the target DNA into sequencing range.
However, certain disadvantages inherent in the methodology of Guo and Wu, supra, limit its usefulness for the large scale sequencing of DNA. For example, this approach depends upon the selection of appropriate restriction enzymes which cleave at restriction sites in close proximity to particular E. coli exonuclease III endpoints, but not within the labeled DNA as this would result in two or more superimposed sequence ladders. The selection of appropriate restriction enzymes generally requires, therefore, the restriction mapping of DNA fragments to identify sites in close proximity to the numerous exonuclease III endpoints. However, the determination of restriction maps tends to be both time consuming and labor intensive. Specifically, restriction mapping to the resolution needed for DNA sequencing involves the digestion of each region of DNA with combinations of 20 or more enzymes to uncover the relative position of restriction sites. This may require over 100 enzymatic reactions followed by numerous electrophoretic separations. Further, significant amounts of DNA are consumed in the mapping process and interpretation of the data generally requires a substantial amount of time.
In addition to the foregoing limitations inherent in current sequencing techniques, the generation of DNA substrate molecules for each 300 to 500 nucleotides to be sequenced is presently required. Assuming no overlapping sequence between substrate molecules, the sequencing of both strands of an entire mammalian genome would, therefore, require the generation of at least 20 million DNA substrate molecules.
A non-ordered approach to sequencing, e.g., shotgun sequencing, would require the generation of 100 to 200 million DNA templates. Although there has been effort directed to automating the steps presently involved in DNA substrate generation, e.g., restriction mapping, preparation of subfragments for subcloning, identification of subclones, growing bacterial cultures, and purifying nucleic acids, it is unlikely that human intervention can be substantially eliminated from the process. Current approaches, therefore, are less than optimal for the large scale sequencing of DNA, particularly sequencing the human genome.
Although the problems enumerated above are not intended to be exhaustive, the limitations inherent in methods presently available for sequencing DNA are readily apparent. Accordingly, there exists a need for an improved method of sequencing DNA that circumvents the need for primer binding sites as well as the need to determine restriction maps. Additionally, there exists a need for an improved method which extends the amount of sequence information obtainable from a DNA substrate, thus substantially reducing the number of DNA substrate molecules required to sequence a given region of DNA. The present invention meets these needs.